Liver cancer develops from chronic liver diseases (chronic hepatitis and liver cirrhosis), and most cases from those with persistent infection of hepatitis virus. In Japan over 95% of liver cancer patients have sustained infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), especially over 80% of those derives from HCV-associates diseases. Generally, patients with hepatitis become more severe in phases in 20-30 years after hepatitis occurrence, and develop liver cancer after progression to liver cirrhosis. Especially, pre-liver cirrhosis phase (fibrosis degree F3) and liver cirrhosis (fibrosis degree F4) are associated with high rates of liver cancer, and it's said that those patients will develop liver cancer within 10 years.
The age of onset is 60 years and over, and more cases in old generations. There are cases to develop in relatively early stage and in lower generations after hepatitis occurrence. But the cause is as yet not well known. In the past, screening using immunological assay of existing tumor markers, AFP (alpha-fetoprotein) and PIVKA-II (protein induced by Vitamin K absence-II), and monitoring by an echo test (ultrasonography) for high risk patients of liver cancer have been regularly performed to detect liver cancer.
For patients who have been suspected to have liver cancer by those testing, the place, size and number of liver cancer are confirmed by diagnostic imaging, more detailed echo, CT (Computed Tomography) or MRI (Magnetic Resonance Imaging) tests. Furthermore liver cancer is finally diagnosed definitely using a pathological test by biopsy, if needed. Liver cancer which has been diagnosed definitely is undergone treatment; hepatectomy, transcatheter arterial embolization (TAE), puncture treatment (percutaneous ethanol injection therapy (PEIT), radiofrequency ablation (RFA) and microwave coagulation therapy (MCT)). However, recurrence rate of liver cancer is extremely high, as virus has been not removed and liver diseases have not been cured even if be treated.
If liver cancer is detected in early stage (below 3 cm in diameter, below 5 cm in diameter and a single or well-differentiated tumors) by the current immunological assay and echo test, and is treated properly, prognosis is relatively good. However, the sensitivity and specificity by existing tumor markers, AFP and PIVKA-II, which are employed for immunological assay, are insufficient. Especially, for liver cancer in early stage both markers have remarkably low sensitivities (see nonpatent literature 1, 2, 3 and 4). Therefore, it is difficult to detect liver cancer in early stage, most cases have already progressed to advanced cancer when detected, and the 5-year survival relative rate of liver cancer patients is very bad in comparison with other cancers (see nonpatent literature 5).
Meanwhile, recent improvement of instruments in an echo test has enabled us to detect liver cancer in early stage, but it is not suited to screening for high risk patients of liver cancer because of some troubles to take around 30 min for a test, to demand technical skills, to depend on performances of instruments and to remain low penetration rate over general hospitals. Furthermore, as it is difficult to examine whole liver, especially parts hidden by other organs and inside of liver completely, there is the risk overlook liver cancer. Thus, there have been no sufficiently satisfactory existing methods for detection of liver cancer, especially that in early stage.
Recent development of molecular biological technology is clarifying characteristics of DNA modification without any genetic changes and any alternations of nucleotide sequences in human cancer cells. Characteristics of changes, mutation, amplification and loss of genes, are well known as representative examples. By recent epigenetical study, there have been reports that particular genes are highly and abnormally methylated in variable cells, especially cancer cells.
In higher eukaryotes, genomic DNA is methylated at only cytosine residues in CpG dinucleotides, the CpG rich region is called as “CpG island” and it is known to exist on around half of whole human genome. Generally, the CpG island inhabits the promoter region, and in that case methylation is strongly concerned in regulation of gene expression. Briefly, in unmethylated cases genes are expressed (mRNA is transcribed), but in methylated cases gene expression is contrarily suppressed through some processes for regulation system. It is known that this regulation of gene expression by methylation is specific to tissue, development, differentiation, disease, sex or aging. Especially, as suppression of gene expression by abnormal high methylation on CpG island is frequently observed in cancer cells or transformed cells, it is considered to be related to carcinogenesis.
Based on those evidences in fundamental study, until now clinical studies with regard to association between abnormal high methylation on CpG island and carcinogenesis have been performed in variable cancers. In liver cancer similar clinical studies have been carried out, and it has been reported that abnormal high methylation on particular genes is observed in tumor tissues of liver cancer patients (see nonpatent literature 6, 7, and 8).
Accordingly, it is inferred that utilization of identification of methylation on particular genes in cancer cells or their DNA fragments for diagnosis is very useful for detection of cancer in early stage. Furthermore, methylation which is considered as one of factors directly relating to carcinogenesis is supposed to develop before cancer occurrence, to alter with progression or malignant alteration of cancer, and to show different profiles from differing patients or clinical conditions. Therefore, Identification of methylation has possibilities to enable to utilize for detection of precancerous lesion, prediction of recurrence after treatment of liver cancer, detection of malignancy of liver cancer and monitoring of progression of liver cancer with time.
However, the previous results of clinical studies are obtained using tumor tissue DNA after confirmed diagnosis for specimens to analyze, and can not meet a screening use for detection of liver cancer in early stage or risk of liver cancer occurrence. An assay using tissue is not suited to a routine testing due to complicated manipulation and very low capacity of sample processing. Furthermore, the sensitivity and specificity are insufficient because of improper selection of genes to analyze and a single gene usage for analysis. Thus, it has been very difficult to utilize identification of methylation on DNA fragments for cancer diagnosis practically.
Meanwhile, minute DNA is circulating in blood, and especially it is known that DNA amount in blood are rising in cancer patients (see nonpatent literature 9 and 10). It is considered to be led by which older cells are destroyed by program death (apoptosis) or necrosis and large quantities of DNA are released from them into blood, while tumor tissue continues to proliferate at abnormal speed. Because DNAs from variable tissues or blood cells as well as DNA which is derived from tumor tissue are included in blood, it has been difficult to discriminate DNA derived from a particular tumor tissue by using conventional technologies. But, to utilize DNA in blood for cancer diagnosis is very promising for practical application of diagnosis.